1. Field of the Invention
The present invention relates to a coryneform bacterium that is able to efficiently produce a heterologous protein by secretion, and a method for secretory production of a heterologous protein.
2. Brief Description of the Related Art
To date, secretory production of heterologous proteins by microorganisms has been reported in Bacillus bacterium (Microbiol. rev., 57, 109-137 (1993)), methanol-assimilating yeast, Pichia pastoris (Biotechnol., 11, 905-910 (1993)), filamentous fungi of the genus Aspergillus (Biotechnol., 6, 1419-1422 (1988) and Biotechnol., 9, 976-981 (1991)), and so forth.
Secretory production of heterologous proteins by coryneform bacteria has also been reported, specifically secretion of a nuclease and a lipase by Corynebacterium glutamicum (henceforth also abbreviated as C. glutamicum) (U.S. Pat. No. 4,965,197 and J. Bacteriol., 174, 1854-1861 (1992)), secretion of a protease such as subtilisin (Appl. Environ. Microbiol., 61, 1610-1613 (1995)), secretion of a protein using signal peptides of cell surface layer proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria (Japanese Patent Laid-open (Kohyo) No. 6-502548), secretion of a fibronectin-binding protein using the signal peptide of PS2 (CspB) (Appl. Environ. Microbiol., 63, 4392-4400 (1997)), secretion of protransglutaminase using signal peptides of cell surface layer proteins PS2 (CspB) and SlpA (also referred to as CspA) of coryneform bacteria (Japanese Patent No. 4320769), secretion of a protein using a variant type secretion system (Japanese Patent Laid-open (Kokai) No. 11-169182), secretion of a protransglutaminase by a variant strain (Japanese Patent No. 4362651), secretion of a protein using a Tat-dependent signal peptide (Japanese Patent No. 4730302), and so forth.
Various proteins have been suggested as proteins which could be produced by secretory production; however, in coryneform bacteria, there are no reports of secretory production of any multimeric protein, such as, for example, antibody-related molecules.
Penicillin-binding protein (PBP) is a generic term which describes proteins that bind with β-lactam type antibiotics, and as a result, inhibit binding with β-lactam type antibiotics. PBPs are generally membrane-binding proteins, and they are considered essential for cell wall synthesis of eubacteria. PBPs are classified as high molecular weight PBPs (HMW-PBPs) or low molecular weight PBPs (LMW-PBPs), according to their molecular weights. HMW-PBPs are further classified as class A high molecular weight PBPs (class A HMW-PBPs), which have both a transpeptidase activity domain for crosslinking peptidoglycan moieties, and a transglycosylase activity domain for forming a polysaccharide chain from disaccharides, and class B high molecular weight PBPs (class B HMW-PBPs) which have only a transpeptidase activity domain.
The findings about PBPs of C. glutamicum are detailed in Mol. Microbiol., 66, 643-57 (2007), Antonie Van Leeuwenhoek, 94, 99-109 (2008), Mol. Microbiol., 9, 97-109 (1993), and J. Biotechnol., 112, 177-193 (2004). In C. glutamicum, at least nine PBP homologues have been found so far. Five of them are HMW-PBPs including two class A HMW-PBPs (PBP1a, PBP1b), and three class B HMW-PBPs (FtsI, PBP2a, PBP2b). It is known that the class A HMW-PBPs of C. glutamicum are responsible for cell extension, and the class B HMW-PBPs are responsible for formation of peptidoglycan of septal walls at the time of cell division.
Cell surface layer proteins are proteins constituting the cell surface layers (S-layers) of bacteria and archaea. As the cell surface layer proteins of coryneform bacteria, PS1 and PS2 (CspB) of C. glutamicum (Mol. Microbiol., 9, 97-109 (1993)), SlpA (CspA) of C. stationis (Japanese Patent Laid-open (Kohyo) No. 6-502548), and so forth are known. Regarding PS2 (CspB), for example, amino acid sequences of CspB homologues of 28 strains of C. glutamicum have been reported (J. Biotechnol., 112, 177-193 (2004)). As described above, signal peptides of cell surface layer proteins of coryneform bacteria are utilized in secretory productions of proteins (Japanese Patent Laid-open (Kohyo) No. 6-502548; Japanese Patent No. 4320769, and so forth).
However, the relationship between the decrease in the activity of a penicillin-binding protein and/or the decrease in the activity of a cell surface layer protein, and the secretory production of a heterologous protein has not been previously reported.